Methods And Products For Delivering Biological Molecules To Cells Using Multicomponent Nanostructures

ABSTRACT

This invention is predicated on the present applicants&#39; discovery that nanostructures comprising discrete regions of different composition can be used to deliver to a biological cell a desired combination of molecules in close proximity. Different molecules can be selectively bonded to discrete regions of different composition in sufficiently close physical relationship to enhance delivery or effectiveness within the cell. The preferred nanostructures are multicomponent nanorods. Important applications include delivery of missing DNA sequences for gene therapy and delivery of antigens or DNA encoding antigens for vaccination.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/875,543, fled Jun. 24, 2004, which claims the benefit of U.S.Provisional Patent Application Ser. No. 60/482,141 filed by Dr. Ali-agerK. Salem et al on Jun. 24, 2003 and entitled “Multifunctional Nanorodsfor Gene Delivery”, which is incorporated herein by reference.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under DARPA/AFOSRcontract number F49620-02-1-0307. The government has certain rights inthis invention.

FIELD OF THE INVENTION

This invention relates to methods of delivering biological molecules tocells and, in particular, to methods of delivering to cells a desiredcombination of biological molecules in close physical proximity. It alsoincludes products for effecting such delivery.

BACKGROUND OF THE INVENTION

The capability of delivering biologically active molecules to plant andanimal cells is of great importance to medicine and genetic research andengineering. In medicine, for example, the development of effectivevaccines requires systems for providing characteristic portions ofinfectious biological entities to immune system cells so that the immunesystem will recognize and fight an infection. When such characteristicportions (antigens) of entities such as viruses, bacteria or even tumorsare appropriately provided, the immune systems identifies the antigensas foreign and stimulates development of immunological countermeasures.One way to provide antigens is to deliver them directly into cells.Another way is to deliver to the cells DNA sequences that encode theantigens.

Gene therapy seeks to introduce additional genetic material (typicallyDNA) into a cell in such a way that the additional genetic material willbe functionally incorporated into the existing genetic material of thecell, For example, there are certain diseases that are caused by theabsence in cells of normally present DNA sequences (genes) needed tomake critical proteins. Gene therapy seeks to alleviate such diseases byproviding the cells with the missing DNA sequences so that the cellsthemselves can provide the critical proteins. To achieve this goal, themissing DNA sequences need to be introduced into cells in such a fashionthat they are functionally incorporated into the genetic material andmechanisms of the cells.

The effectiveness of an active biological molecule in a cell often canbe enhanced by the presence of one or more additional differentmolecules. For example, there are molecules, called adjuvants, that willincrease the likelihood that an antigen will be recognized as anappropriate target for immunological countermeasures. As anotherexample, there are also molecules that will interact with cell receptorsand increase the likelihood of incorporation into the cell. Suchenhancing molecules, however, typically must be close to the activemolecule in order to enhance its effectiveness.

Conventional approaches to delivering biological molecules to cellsleave much to be desired. The common approach to gene therapy is basedon the fact that viruses have evolved to inject genetic material into acell and use the cell's genetic machinery to replicate the viral geneticmaterial. Appropriate modification of the virus might eliminate itsharmful features and redirect a viral vector to deliver desirablegenetic material into the cell. However virus vectors often generatecounterproductive host immune responses and present a risk of killinginfected host cells (cytotoxicity).

Other delivery approaches that have been suggested include the use ofcarriers comprising liposomes, pollaners and gold nanoparticles. Theyhave not, however, achieved notable success in efficiently incorporatingnew genetic material or in making more effective vaccines. Accordinglythere Is a need for improved methods and products for deliveringbiological molecules to cells.

SUMMARY OF THE INVENTION

This invention is predicated on the present applicants' discovery thatnanostructures comprising discrete regions of different composition canbe used to deliver to a biological cell a desired combination ofmolecules, including at least one biological molecule, in closeproximity. Different molecules can be selectively bonded to discreteregions of different composition in sufficiently close physicalrelationship to enhance delivery or effectiveness within the cell. Thepreferred nanostructures are multicomponent nanorods. Importantapplications include delivery of missing DNA sequences for gene therapyand delivery of antigens or DNA encoding antigens for vaccination, andsimultaneous delivery of interacting medicines in specific proportionand close proximity,

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The advantages, nature and various additional features of the inventionwill appear more fully upon consideration of the illustrativeembodiments now to be described in detail in connection with theaccompanying drawings. In the drawings:

FIG. 1 is a schematic block diagram of a method of delivering biologicalmolecules to cells in accordance with the invention.

FIG. 2 is a schematic diagram illustrating functionalization ofmulticomponent nanostructures. In FIG. 2 a nanorods are incubated withthe 3-[(2-aminoethyl)dithio] propionic acid (AEDP) linker. Thecarboxylate end group binds to the nickel segment. The disulfide linkageat the center acts as a cleavable point within the spacer promoting DNArelease within the reducing environment of the cell. In FIG. 2 bplasmids are bound by electrostatic interactions to the protonatedamines presented on the surface of the nickel segment. In FIG. 2 ccalcium chloride compacts the plasmids encoding the luciferase or GFPreporter genes; and in FIG. 2 d rhodamine-conjugated tansferrinpresenting sulfhydryl groups is selectively bound to the gold portion ofthe nanorods.

FIG. 3 shows microphoto images pertaining to functionalizedmulticomponent nanostructures. FIG. 2 a is a visible light image of dualfictionalized 200 nm long AuNi nanorod. FIG. 3 b fluorescence image ofthe rhodamine-tagged (543/570 nm) transferrin on the Au segment. FIG. 3c is a fluorescence image of the Hoechst stained (350/450 nm) plasmidson the Ni segment; and FIG. 3 d is a fluorescent overlay image combiningFIGS. 3 b and 3 c.

FIG. 4 shows microphoto images of cells transfected in accordance withthe method of FIG. 1. FIG. 4 a presents stacked laser scanning confocalmicroscope images of a live HEK293 cell (red/633 nm, green/543 nm).Rhodamine (633 nm) identifies the sub-cellular location of the nanorodswhilst GFP expression (543 nm) provides confirmation of transfectionthroughout the cell. FIGS. 4 b and 4 c are, orthogonal sections thatconfirm the nanorods are within the cell. FIG. 4 d shows confocalmicroscope stacked images, of a live HEK 293 cell stained withLysotracker Green identifying the location of the nanorods (Rhodamine)in relation to acidic organelles in both orthogonal sections (FIGS. 4 eand 4 f).

FIG. 5 presents scanning electron microscope images of cells transfectedin accordance with the method of FIG. 1. FIG. 5 a is a SEM image ofHEK293 cells after Ih incubation with 200 nm Au/Ni nanorods. FIG. 5 b isa back-scattering SEM image of 200 nm Au/Ni nanorods after 4 hincubation showing the nanorods beneath the surface of the cell. FIG. 5c is a TEM cross-sectional image showing the intra-cellular location ofthe nanorods after 4 h incubation, and FIG. 5 d is a, SEM image of 200nm long nanorods after 4 h incubation.

FIG. 6 is a set of histograms summarizing results of transfectionexperiments. FIG. 6 a shows percentage of GFP expression (area of cellsfluorescing/total cell area) and FIG. 6 b shows luciferase expressionof: 1. nanorod-plasmid complex, 2. nanorod-plasmid/transferrin complex,3. nanorod-plasmid/transferrin complex incubated with 100 micromoleschloroquine, 4. Lipofectamine (positive control) and 5. naked DNA(negative control).

FIG. 7 is a graphical illustration of ovalbumin-specific antibodyresponses in C57BL/6 mice immunized with various antigen or plasmidnanorod and gold particle formulations. C57BL/6 mice were immunized withcontrol plasmid (no insert) bound to nanorods, ovalbuminantiegen-nanorod formulation, ovalbumin antigen-gold particleformulation, pcDNA3-OVA7-nanorod formulation, pcDNA3-OVA7-gold particleformulation and ovalbumin antigen/control pcDNA3 (no insert)nanorodformulation via a gene gun. Serum samples were obtained from immunizedmice 21 days after the initial vaccination. The presence of theovalbumin-specific antibody was detected by ELISA using serial dilutionof sera. The results from the 1:1000 dilutions are presented showing themean absorbance (A450 nm)±SE.

FIG. 8 graphically illustrates ovalbumin-specific CD8+T-cell precursorsin C57BL/6 mice immunized with various antigen or plasmid-nanorod andgold particle formulations. C57BL/6 mice were immunized with controlplasmid (no insert) bound to nanorods, ovalbumin antigen-nanorodformulation, ovalbumin antigen-gold particle formulation,pcDNA3-OVA7-nanorod formulation, pcDNA3-OVA7-gold particle formulationand ovalbumin antigen/control pcDNA3 (no insert)-nanorod formulation viaa gene gun. For vaccinated mice, 2 μg of DNA or antigen/mouse were giventwice. Splenocytes were harvested 7 days after the last DNA /antigenvaccination. Flow cytometry analysis: Splenocytes from vaccinated micewere cultured in vitro with the ovalbumin antigen overnight and werestained for both CD8 and intracellular IFN-. The number of IFN-secreting (CD8+ T-cell precursors in mice immunized with antigen orplasmid-nanorod and gold particle formulations were analyzed by flowcytometry. The number of CD8+IFN-+double-positive T cells in 3×10⁵splenocytes are represented by the quadrant in the upper right corner.

FIG. 9 schematically illustrates formation of three component nanowires.

FIG. 10 schematically shows a general approach for selectivederivatisation of Au/Ni/Pt nanowires; and

FIG. 11 is a set of micrographs illustrating 3 component nanostructures.FIG. 11 a is a back-scattering SEM image of nanowires showing integrityof Au, Ni and Pt segments. The image confirms platinum segments arelonger than Ni and Au segments. FIGS. 11 b and 11 c light andfluorescence microscope images of Au/Ni/Pt nanowires functionalized withBIC and Rhodamine Red-12-dodecanoic acid (Ex 570, Em 590). Confirmationof selective derivatisation of Au/Ni/Pt nanowires with BIC, RhodamineRed-12-dodecanoic acid and Marina Blue-1-undecane-thiol is observed bylight microscope images (FIG. 11 d) and fluorescence microscope imagesFIGS. 11 e-11 g. FIG. 11 d is a light microscope image of afunctionalized Au/Ni/Pt nanowire. FIG. 11 e shows the fluorescence fromthe Marina Blue-1-undecane-thiol (Ex 365, Em 460) bound to the goldsegment. FIG. 10 e shows the fluorescence from the RhodamineRed-12-dodecanoic acid (Ex 570, Em 590) from the nickel segment, andFIG. 11 g is a fluorescent overlay image combining 10 e and 10 f.

It is to be understood that these drawings are for illustrating theconcepts of the invention and, except for the graphs, are not to scale.

DETAILED DESCRIPTION

Referring to the drawings, FIG. 1 is a schematic block diagram of amethod for delivering biological molecules to cells in accordance withthe invention. As shown in Block A, an initial step is lo provide amulticomponent nanostructure comprising at least two discrete regions ofrespectively different materials. The term “nanostructure” as usedherein refers to structures having maximum dimensions in at least twodimensions that are substantially smaller than the diameter of a cell sothat the structures may enter a cell without destroying itsfunctionality. Typically the nanostructure has two maximum dimensions ofless than about 500 nanometers and preferably less than about 200nanometers. The maximum third dimension is also preferably less than thediameter of a cell so that the nanostructure can be incorporated in thecell, but it can be greater (into the micrometer range) and stilltransfect a cell. Useful multicomponent nanostructures have at least twodiscrete regions large enough to bind respective biological moleculesbut positioned sufficiently close together that both molecules can bedelivered into the same cell at the same time.

The inventive method can use multicomponent nanostructures in a widevariety of sizes and shapes including multicomponent nanorods,nanowires, nanotubes, nanoscale bars, nanodisks, nanoscale ovals,nanoscale parallelpipeds and multicomponent nanoparticles of regular orirregular shape. Multicomponent nanostructures with any one of a widevariety of shapes, sizes and material combinations can be fabricated bytechniques well known in the art, as by depositing successive nanolayerson a removable substrate, patterning the layers by nanoimprintlithography, and removing the substrate. Further details concerningnanoimprint lithography can be found, for example, in U.S. Pat. No.6,309,580 issued to Stephen Chou on Oct. 30, 2001, which is incorporatedherein by reference.

The preferred multi component nanostructures are nanorods or nanowirescomprised of discrete segments of respectively different materials (SeeFIG. 2). Such segmented rods or tubes can be fabricated, for example, byelectro-depositing successive layers of different metals in a nanoporousmatrix material and removing the matrix material, as by selectivelydissolving in acid or base.

The next step shown in Block B is to attach one or more molecules ofdifferent materials to the respectively different discrete regions ofthe nanostructure. In essence, each different molecule is provided witha chemical group that selectively bonds to a respectively differentmaterial of the multicomponent nanostructure. The preferred moleculesfor attachment are biological molecules. The term “biological molecules”as used herein includes, without limitation, molecules of geneticmaterial (DNA and RNA), molecules of materials that activate cellreceptors (external or internal), antigens or their genetic material,and materials that enhance the incorporation of genetic material orstimulate the immune response. The term also includes molecules ofmedications that are active at the cellular level, and especiallydifferent medications that have a synergistic effect when deliveredtogether. Thus, for example, molecules to stimulate cell receptors canbe selectively bonded to a first material segment of a nanorod and a DNAsequence can be selectively bonded to a second material segment. Asanother example, DNA encoding an antigen can be selectively bonded to afirst segment and an immune system stimulating adjuvant molecule can bebonded to a second segment, and an antigen can be bonded to a yet thirdsegment. Exemplary of useful RNA biological molecules is siRNA that canbe used to silence undesirable genes. Thus a multicomponentnanostructure could contain RNA to silence a defective gene and DNA toprovide the correct gene. An example of synergistic medications thatcould be simultaneously delivered by multicomponent nanostructuresinclude Taxol and Discodermolide.

The third step, Block C, is to deliver the nanostructure and its bondedmolecules to biological cells. The method of delivery may depend on thelocation and type of cells. For delivery to somatic cells, the preferredapproach is to use a nanostructure including a bonded biologicalmolecule to stimulate cell receptors that will take the structure intothe cell. The nanostructures can be introduced, as by pneumaticinjection, into desired tissues and stimulated cell receptors willfacilitate their intake into cells. For dendritic cells located near thesurface of the body, the nanostructures may be injected directly intothe cells as by pneumatic pressure. Deeper penetration into somaticcells may be achieved by orienting nanotubes so that their cylindricalaxes are aligned approximately perpendicular to the target tissue at thepoint of injection.

As will be illustrated in the exemplary embodiments described hereinbelow, a major advantage of this method is the ability to simultaneouslyprovide specific combinations of biological molecules in close adjacencywhere they can interact to produce more effective biological results,e.g. more effective incorporation in the cell, an enhanced immuneresponse, or a more effective combination of medicines.

The invention can now be more clearly understood by consideration of thefollowing examples.

EXAMPLE 1

Delivery of Genetic material For Gene Therapy

The goal of gene therapy is to introduce foreign genes into somaticcells to supplement the defective genes or to provide additionalbiological functions. Gene transfer (“transfection”) can be achievedusing either viral or synthetic non-viral delivery systems (“vectors”).While viral vectors exhibit high efficiency, synthetic transfectionsystems provide several advantages including ease of production andreduced risk of cytotoxicity and immune response. Much of the poortransfection efficiency of non-viral vector stems from the difficulty ofcontrolling their properties at the nanoscale. One aspect of the presentinvention is a novel non-viral delivery system based on nanostructuresthat can simultaneously bind compacted DNA plasmids and target cellreceptors for enhanced internalization. The present example demonstratesthe potential of this system to deliver genetic material with precisecomposition and size control.

Achieving efficient gene delivery into a target cell population ortissue without causing associated toxicity is critical to the success ofgene therapy. To this end, both viral and non-viral vectors have beenextensively investigate. Although viral vectors such as adenovirus,lentil virus, influenza virus, and adeno-associated virus are efficientin transfecting cells, their toxicity and immunogenicity remain severelimitations.

As alternatives to viruses non-viral vectors such as liposomes andpolymers have been increasingly studied to overcome this long-termsafety issue. In contrast, inorganic gene carriers have received limitedattention in the gene therapy community. Gold nanoparticles with boundDNA are used in particle bombardment-mediated gene transfer (“gene guntechnology”). While this gene gun technology may be effective intransfecting cells in the skin for genetic immunization, it has limitedutility in general gene transfer applications involving internal organtransfection.

To be effective, non-viral vectors must gain entry into the target cellsand then release the condensed plasmid into the cytoplasm fortranslocation into the nucleus. To date, particle-based vectors' havebeen formulated by using polycationic polymers or lipids to condense DNAinto nano-complexes that can be internalized by cells. The size of thesenano-complexes is typically difficult to control and widely dispersed.Targeting ligands can be conjugated to the carrier or complexes eitherpre- or post- complexation with the DNA from the complexes may alsobecome a rate-limiting step. To optimize these different aspects indesigning an effective non-viral gene delivery system has been a majorchallenge in the field.

The possibility of achieving control of size and composition byinorganic synthesis has prompted us to evaluate the potential ofmulti-segment metallic carriers in gene delivery. In this example, wedemonstrate the novel properties of bi-functional Au/Ni nanorods in genetransfer. Deposition of the AuNi nanorods was achieved by templatesynthesis. This technique involves electrochemical deposition into anon-conducting membrane having an array of cylindrical pores and hasbeen used for the synthesis of a wide range of materials and structures.Template synthesis is preferred over other techniques because it iseasily adapted for the deposition of multiple sub-micron segments.Furthermore, template synthesis can produce large quantities ofmonodisperse nanorods, and properties such as aspect ratio can becontrolled in a systematic way.

Referring to FIG. 2, the nanorods 20 were fabricated byelectrodeposition into an Al₂O₃ template (Anodisc, Whatman) with a porediameter of 100 nm. An evaporated silver film on one side of thetemplate served as the working electrode in a three-electrodeconfiguration. Ak thin layer of silver was electrodeposited into thetemplate from 50 mM KAg(CN)₂, 0.25 M Na₂CO₃ buffered to pH 13 at apotential of −1.0V (Ag/AgCl) and Ni segments 21 were deposited from asolution of 20 g L⁻¹ NiCl₂·6H₂O, 515 g L⁻¹ Ni(H₂NSO₃)₂·4 H₂O), 20 gL⁻¹H₃BO₃ buffered to pH 3.4 at a potential of −1.0 V (Ag/AgCl) to ensureeasy release of the nanorods from the template. The Au segments 22 weredeposited from a commercial gold plating solution (Technic Inc.) at apotential of −1.0V (Ag/AgCl) and the Ni segments 21 were deposited froma solution of 20 gL⁻¹ NiCl₂·6 H₂O, 515 gL⁻¹ Ni(H₂NSO₃)₂·4 H₂O), 20gL⁻¹H₃BO₃ buffered to pH 3.4 at a potential of −1.0 V (Ag/AgCl). Thesilver layers were dissolved in 70 vol % nitric acid and the aluminatemplate was then dissolved in 2 M potassium hydroxide. The nanorods 20were washed repeatedly using 2 M potassium hydroxide, de-ionized waterand ethanol. The nanorods were 100 nm in diameter and 200 nm in lengthwith 100 nm gold segments and 100 nm nickel segments.

Using molecular linkages that bind selectively to either gold or nickel,we have attached DNA 23 and a cell-targeting protein 24, transferrin, tothe different segments, as shown schematically in FIG. 2. Transferrinwas one of the first proteins to be exploited for receptor-mediatedendocytosis of the transferrin-iron complex. The transferrin 24 wasbound to the gold segments 22 of the nanorods 20 through a thiolatelinkage (not shown), by converting a small proportion of the primaryamine groups of transferrin to sulfhydryl groups. A rhodamine tag (notshown) on the transferrin provided a mechanism for confirmation ofinternalization and intracellular tracking of the nanorods.

DNA 23 was bound to the nickel segments 21 by suspending the dualcomponent nanorods in a 0.1 M solution of 3-[2-aminoethyl)dithio]propionic acid (AEDP). The carboxylic acid terminus of AEDP binds to thenative oxide on the nickel segments This resulted in the surfacepresentation of primary amine groups spaced by a reducible disulfidelinkage 25. Plasmids encoding the firefly luciferase (pCMV-luciferaseVR1255_C) with 6.413 kbt driven by the cytomegalovirus (CMV)promoter/enhancer (luciferase-plasmid) or plasmids encoding the GFPmut1variant (PEGFP-C1) with 4.7 kb driven by the SV40 early promoter(GFP-plasmid) were conjugated to the AEDP bound to the nickel segments21 of the nanorods 20 at pH 5.7. The plasmid concentration, determinedfrom absorbance spectroscopy, was about 4×10¹² molecules cm⁻².

To further compact: the DNA bound to the nanorods for more efficientcell entry and protection of the DNA from enzyiatic degradation, thenanorods were incubated in 2M CaCl₂ after excess non-bound plasmids hadbeen removed. Ca²⁺ has a high affinity to DNA (K_(d) of 1.1×10⁻³M⁻¹),forming CaPO₄ complexes with the nucleic backbone to providestabilization and compaction to the DNA structure.

Confirmation of the selective binding of transferrin and plasmid wasobtained by fluorescence microscopy. Since the 200 nm long nanorodscannot be seen by optical microscopy, these experiments were performedon 20 micron long and 100 nm diameter nanorods with Ni and Au segmentsof equal length.

FIG. 3 shows uniform red fluorescence from the rhodamine-taggedtransferrin on the gold segments and uniform blue fluorescence from theHoechst, which selectively binds to the DNA conjugated to the nickelsegments.

To evaluate the gene delivery potential of these dual functionalizedAu/Ni nanorods, in vitro transfection experiments were performed on theHuman Embryonic Kidney (HEK293) mammalian cell line with the GFP andluciferase reporter genes, respectively. For transfection, the nanorodswere incubated with HEK293 cells at a dosing level (4.4×10⁻⁵ mg mL⁻¹)significantly below the cytotoxicity (LD50 ) value for 4 hours inOpti-MEM cell culture medium (Gibco BRL, Rockville, Md.). Followingwashing, cells were further incubated in serum-containing media for twodays.

FIG. 4 shows confocal microscopy sections of cells followingtransfection. FIG. 4 a shows the characteristic green fluorescence fromthe GFP expressed by the cells as a result of tranfection. Superimposedon the GFP emission is the red emission from the rhodamine conjugated tothe Au segments of the nanorods. The orthogonal sections show clearlythat the nanorods are located in the cell. FIG. 4 d shows fluorescenceimages from cells after 4 h incubation that have been stained withLysotracker green revealing that the nanorods are located in or aroundacidic organelles.

The uptake of the nanorods by HEK293 cells is shown in the scanningelectron microscope images in FIGS. 5 a and 5 b, after 1 and 4 hoursincubation, respectively. Transmission electron microscope images (FIG.5 c) showed that nanorods were located in vesicles or the cytoplasm butnot the nucleus. This suggests that transfection is due to plasmidsreleased or cleaved from the nanorods prior to nuclear entry. Incontrast, 20 μm long nanorods were found only partially internalizedafter 4 hours (FIG. 5 d) presumably because of size constraints.

To further understand the transfection mechanism, a series ofexperiments were undertaken to compare the two-component nanorods withand without transferrin and chloroquine. Chloroquine is an endosomolyticagent widely used to promote escape of the sequestered complexes fromendosomal into cytoplasmic compartments.

FIG. 6 summarizes the transfection experiments. A significantly higherfraction of cells expressed GFP when transfected with plasmid-nanorodsthan with naked DNA, which was <3%. Comparing with the luciferaseplasmid, transfection by nanorods shows a 255-fold higher expressionthan naked DNA. Nanorods with transferrin produced 22% of GFP-positivecells, 2 times higher than those with transferring; the enhancement is3.4 times for luciferase expression level. Addition of chloroquine tonanorods with transferrin further improved GFP expression to 27% ofpositive cells, and increased the luciferease expression level by afactor of 1.9. The fact that chloroquine enhances transferrin-mediatedtransfection suggests that receptor-mediated endocytosis is involved.Chloroquine may also enhance transfection by protecting against DNAdegradation.

To confirm that transfection was due to intracellular rather thanextracellular release of plasmids, nanorods complexed with theluciferase-plasmid were incubated in serum-containing media. Thesupernatant was removed at various time points from 15 minutes to 4hours and used to transfect the HEK293 cells. In all cases nosignificant transfection above background could be detected in thesesamples. These data confirm that the transfection detected is a resultof the intracellular released plasmids from the 200 nm nanorods. Furtherdetails concerning the methods and materials of Example 1 are set forthin Appendix A attached hereto.

In summary, this example demonstrates a new approach for gene deliveryusing multi-segment nanorods. Using molecules with end-groups thatselectively bind to different metals, specific functionalities can beintroduced to individual segments in the nanorod. Here we have useddifferential binding to attach plasmids and a cell-targeting protein tospatially separated regions of the delivery system. This approach can beextended to include other components that allow additionalfunctionalities to be introduced. For example, an additional segmentcould be used to bind an endosomolytic agent. In addition to componentsthat allow selective binding, other functions can also be exploited. Forexample, an external magnetic field can be used to manipulate nanorodswith magnetic segments. In addition, the introduction of segments ofsemiconductor materials can be used to trick individual nanorods throughtheir characteristic absorbance or photoluminescence. The ability toconfigure different segments in varying combinations and with differentsegment lengths can also be used to barcode individual nanorods. Theseproperties can be exploited to externally control gene delivery in vivo.Thus, this versatile synthetic gene delivery system may help realize thepotential of non-viral gene therapy.

EXAMPLE 2 Vaccinations

The goal in genetic vaccinations is to encode cells to transientlymanufacture antigens that are subsequently taken up by macrophages ordendritic cells (key antigen presenting cells or APCs). APCs processthese antigens via class I or class II pathways where they bind to majorhistocompatibility complexes that present the antigen on the surface ofthe APCs. These APCs then move to the lymphoid organs where Tlymphocytes that scavenge the surfaces of the APCs become stimulated torespond against the antigen presented. When, for example, the encodedantigen is tumor specific a strong CD8+ and CD4+ T-cell and antibodyresponse can be generated for protection and prevention against thattumor The inorganic nanorod vectors described herein can generate strongbut transient transgene expression when bombarded into skin, which hasnatural abundance of antigen presenting cells. These nanorods thereforehave potential for vaccination applications. In contrast to otherinorganic non-viral vectors, these nanorods can be engineered withdifferent functionalities in spatially defined regions, which lead tothe potential for precise control of antigen: adjuvant ratios and thepossibility of stimulating multiple immune responses. However, beforethese unique nanorod properties can be exploited for furtherdevelopment, it is essential to ensure that the nanorods can generate astrong versatile immune response in vivo.

In this example, we evaluate the CD4+ antibody and CD8+ T-Cell responsesfrom particle bombardment of nanorods delivering the model antigenovalbumin or plasmids encoding ovalbumin. Ovalbumin is involved in anumber of conditions related to children. For example, children withcystic fibrosis display higher anti-ovalbumin antibodies. Ovalbuminantibodies are also observed in kidney diseases such as nephropathy.Children with insulin dependent diabetes mellitus show elevated immuneresponses to both β-lactoglobulin and ovalbumin, which may be associatedwith the progression of the disease.

The nanorods were fabricated by electrodeposition into an Al₂O₃ template(Anodisc, Whatman) with a nominal pore diameter of 100 nm. An evaporatedsilver film on one side of the template served as the working electrodein a three-electrode configuration. A thin layer of silver waselectrodeposited into the template to ensure easy release of thenanorods from the template. Au segments were deposited prior to nickelsegments to prevent erosion of the nickel layers during silver removal.The silver layers were dissolved in 70 vol % nitric acid and the aluminatemplate was then dissolved in 2 M potassium hydroxide. The nanorodswere 1.6 μm in length by 170 nm in diameter with 800 nm length goldsegments and 800 nm length nickel segments.

Confirmation of deposition of the nickel and gold segments was seen byback-scattering SEM. Using chemical moieties that bind selectively toeither gold or nickel, we attached plasmids of the antigen ovalbumin, tothe different segments as described previously. A small proportion ofthe primary amine groups of ovalbumin were converted to sulfhydrylgroups. The ovalbumin was then bound to the gold segments of thenanorods through a thiolate linkage. Electrostatic interactions wereused to bind DNA to the nickel segments by suspending the dual componentnanorods in a 0.1 M solution 3-[(2-aminoethyl)dithio] propionic acid(AEDP). The carboxylic acid terminum of AEDP binds to the native oxideon the nickel segments. This results in the surface presentation ofprimary amine groups spaced by a reducible disulfide linkage. In thereducing environment of the cell, the disulfide linkage between theplasmid and the nanowire is cleavable, enhancing release of the plasmid.In this example, plasmids encoding ovalbumin (pcDNA3-OVA7) or controlplasmids with blank inserts (pcDNA3) were utilized. Previous UV-visiblespectroscopy calibration measurements (260 nm) of DNA binding to thenanowires provided an average surface coverage of 4×10¹² molecules/cm².For condensation of the plasmids bound to the nanowires, a CaCl₂solution was added to the n-nanowire-plasmid formulations. Ca²⁺ has ahigh affinity to DNA (K_(d) of 1.1×10⁻³M-⁻¹, forming CaPO₄ complexeswith the nucleic backbone to provide stabilization and compaction to theDNA structure.

To evaluate the genetic vaccination potential of these nanorods, CD4+antibody responses from the bloodstream and CD8+ T-cell responses fromthe spleen were measured from C57BL/6 mice vaccinated with thenanorod/plasmid or nanorod/antigen formulations. In addition, wecompared these responses to the industrially optimized gold particleformulations as analogous responses are essential for the futuredevelopment of these nanorods in clinical applications. Forantigen/microcarrier formulations, the gold particles generated a 7-foldhigher CD8+ T-cell response that the nanorods. In contrast, for the CD4+antibody response, the nanorods produced a 7-fold higher response incomparison with the 1.6 μm gold particles (FIGS. 7 and 8). To evaluatethe benefit of the nanorods multifunctionality, pcDNA3 was bound to thenickel segments of the nanorods in conjunction with the ovalbumin-SHantigen on the gold segments. In control experiments, pcDNA3 bound tothe nanorods alone generated very low or no CD4+ antibody and CD8+T-cell responses. However, co-addition of pcDNA3 and the ovalbuminantigen on the nanorods generated a significant 8-fold increase in theCD8 response in comparison to the nanorods bound to the ovalbumin alone.This increase is likely to be due to a role of the CpG motif in thepcDNA3 acting as a strong immunostimulatory adjuvant to the ovalbuminantigen thus enhancing the overall CTL immune response. The nanorodsability to deliver the CpG motif and the antigen to the same cell isessential for generating a stronger immune response. For example, Babuikand colleagues have shown that in pigs, administration of CpG ODN andHBsAg vaccine in separate sites of the sante muscle did not show anenhanced antibody response compared to administration of the HBsAgvaccine alone, whereas administration of CpG ODN with the HBsAg vaccinesignificantly enhanced the antibody responses.

Delivering plasmics encoding ovalbumin by both nanorods and goldparticles generated stronger CD4 and CD8 responses than the ovalbuminantigen alone Gene gun delivery of antigens can directly enter and primedendritic cells, but the delivery of plasmids encoding the antigenprobably enhances the overall response because in addition to directpriming of dendritic cells, keratinocytes also become transfected. Thekeratinocytes then produce antigens that, once released, cross-primemore dendritic cells thereby enhancing overall immune response. Furtherdetails concerning the methods and materials of Example 2 are set forthin Appendix B hereto.

In summary, this example that nanorod based vaccines generate strongCD4+ antibody and CD8+ T-cell responses and therefore have significantpotential for further development in vaccination applications. Wecontemplate that aligning the nanorods within the cartridges to produce“arrow” like delivery will allow us to achieve greater depths ofpenetration in particle bombardment than the gold particles. Advantagesto this would include transfecting both skin and the subcutaneoustissues for pressure modulated control over sustained or transientexpression of genes and greater depths of penetration at lowerpressures. The ability to add new components to the nanorods such asadjuvants and/or cytokines in controlled ratios will allow us togenerate stronger immune responses than single component particles asdemonstrated in this example using the CpG motif from the pcDNA3 as animmunostimulatory adjuvant to the antigen. In addition, the ability toengineer and add extra segments to the nanorods will allow for thepossibility of delivering multiple agents such as RNA, antigens and DNAto the same cell for the stimulation of multiple immune responses.

EXAMPLE 3 Delivery of Multiple Active Molecules

This example demonstrates the selective derivatization of three segmentAu/Ni/Pt nanowires using metal specific ligands. By taking advantage ofthe individual metal segments' affinity to unique functional groups, weshow that Au/Ni/Pt nanowires can be functionalised with a thiol linkageon the gold segments, an isonitrile linkage on the platinum segment anda carboxylate linkage on the nickel segment. Selective functionalisationof the Au, Ni and Pt segments is achieved by first functionalizing theNi segment with carboxylic acid terminated ligands and the Au and Ptsegments with an isonitrile terminated ligand. Carboxylic acids havebeen found to bind to nickel surfaces at an adduct formation constant of6±5×10⁶ M⁻¹. Isonitrile groups are reported to form monolayers on bothAu and Pt surfaces. The isonitrile groups on the Au surface can then beselectively substituted with thiol terminated ligands.

The formation of three component nanowires is shown in FIG. 9, Au/Ni/Ptnanowires 90 are fabricated by electrodeposition into an Al₂O₃ template91 (Anodisc, Whatman) with a nominal pore diameter of 100 nm. Anevaporated silver film 88 on one side of the template serves as theworking electrode in a three-electrode configuration. A thin layer ofsilver 89 is first electrodeposited from 50 mm KAg(CN)2 and 0.25 MNa₂CO₃ buffered to pH 13 at −1.0 V (Ag/AgCl) in order to ensure easyrelease of the nanorods from the template. The Au segments 92 of thenanowires are deposited from a commercial gold plating solution(Technic) at −1.0 V (Ag/AgCl), and the Ni segments 92 are deposited froma solution of 20 g L⁻¹ NiCl₂·6H₂O, 515 g L⁻¹ Ni(H₂NSO₃)₂·4 H₂O), 20 gL⁻¹H₃BO₃ buffered to pH 3.4 at a potential of −1.0 V (Ag/AgCl). The Ptsegments 94 are deposited from a solution of 0.015M of (NH₄)₂·PtCl₆ and0.2M Na₂HPO₄·7H₂O at −0.4 V (Ag/AgCl). The gold segments 92 aredeposited before the nickel segments 93 in order to ensure that thenickel segments 93 are not etched by the nitric acid during removal ofthe silver, and the platinum segments 94 are deposited after the nickeland with longer length segments to clearly differentiate the segmentfrom the gold. The silver layers (88, 89) are dissolved in 70 vol. %nitric acid and the alumina template 91 is then dissolved in 2 M KOH.The nanowires 90 are washed repeatedly using 2 M KOH, de-ionized water,and ethanol. The nanowires 90 are on average 170 nm +/− 23 nm indiameter and 8-10 μm in length.

Confirmation of the integrity of the three segments is observed byback-scattering SEM (FIG. 11 a). Collection of the nanowires bycentrifugation at 8000 rpm often results in bending of the nanowires, inparticular at the junctions of the segments. Magnetic collection of thenanowires results in significantly reduced bending but the remnantmagnetized state of the nickel segments produces aggregated nanowiresthat reduce the efficiency of selective derivatization and leads togreater difficulty in subsequent imaging of single nanowires.

FIG. 10 schematically illustrates the functionalization of the nanowires90. In the first step of the functionalization (FIG. 10 a), Au/Ni/Ptnanowires (≈10⁹ mL⁻¹) are suspended in 2 ml of ethanol containing 2 mM12-amino-dodecanoic acid (Aldrich) and 2 mM 1-butane isocyanide (BIC).The suspension is agitated using rotation for 24 hours. The nanowires 90are then washed using repeat centrifugation and resuspension cyclesusing ethanol. The nanowires are then reacted with 3.84 mg Rhodamine Redsuccinimidyl ester (Molecular Probes) in 5 mL of a 50:50 mixture of pH8.5 sodium tetrahorate buffer and dimethylsulfoxide (DMSO) overnightunder an argon blanket at room temperature (FIG. 10 b). The succinimidylester reacts rapidly in the presence of primary amine groups producing astrong amide bond between the self-assembled monolayer molecules and thefluorophore. The nanowire suspension is then sonicated for 1 hour,followed by washes with DMSO, water and ethanol. For microscopicimaging, nanowires are spin coated onto a glass coverslip rotating at2500 rpm for 15 secs.

FIGS. 11 b and 11 c show light microscope and fluorescence microscopeimages of the functionalized nanowires. Fluorescence from the rhodamine(Ex 570, Em 590) is predominantly localized to the Ni segment.Significantly weaker fluorescence is also observed on the Au and Ptsections. This is most probably due to physisorption between thehydrophobic rhodamire red flurophore and the hydrophobic BICfunctionalised Au and Pt sections. Carboxylic acids have been reportedto bind weakly to Au surfaces. The contrast between the weakfluorescence on the Au/Pt segments and the strong fluorescence on the Nisegment indicates that the BIC has preferentially bound to the Au/Ptsurfaces significantly blocking carboxylic acid binding. Note thatwhilst the quenching of fluorescence molecules proximate to metalsurfaces has been previously reported fluorophores bound to nanowiresremain sufficiently detectable to identify selective functionalization.

Referring back to FIG. 10 the nanorods are next suspended in 2 ml of 2mM 11-amino-1-undecanethiol (Dojindo) in ethanol and mixed usingrotation for 24 hours (FIG. 10 c). The nanowires are washed with ethanolusing repeat centrifugation and resuspension cycles. The nanowires arethen reacted with 3.67 mg Marina Blue succinimidyl ester (MolecularProbes) in 5 mL of a 50:50 mixture of pH 8.5 sodium tetraborate bufferand DMSO overnight under an argon blanket at room temperature (FIG. 10d). The nanowire suspension is then sonicated for 1 hour, followed bywashes with DMSO water and ethanol

FIG. 11 d-11 g show light microscope and fluorescent microscope imagesof the tri-functionalized nanowires. FIG. 11 e shows that fluorescenceobserved from the Marina Blue-1-undecanethiol (Ex 365, Em 460) isspecifically from the gold segment.

FIG. 11 f shows that fluorescence from the rhodamine (Ex 570, Em 590) isstill emanating from the Ni segment. Either very weak fluorescence (Ex365, Em 460) or no fluorescence at all is observed from the longer Ptsections functionalised with BIC. This suggests that the carboxylic acidhas retained its binding affinity to the Ni, whilst the thiol terminatedmolecules have displaced isonitril groups on the Au segment but not thePt section. Surface engineering Au and Pt with BIC first followed bythiol displacement on the Au segment is preferential because of thereported ability of the BIC molecules to prop up the thiol terminatedmolecules in the upright orientation.

In control experiments, Au/Ni/Pt nanowires are functionalized with BICand 12-amino-dodecanoic acid followed by treatment with Rhodamine Redsuccinimidyl ester. when the wires are then exposed to 1-decanethiol,fluorescence is observed only on Ni segments. Similarly, when thenanowires are functionalized with BIC and palmitic acid, followed byexposure to 11-amino-1-undecanethiol, subsequent treatment with Marinablue succinimidyl ester results in fluorescence predominantly observedon the Au sections.

In summary, this example demonstrates selective derivatization of threecomponent Au/Ni/Pt nanowires using metal specific surface chemistries.The ability to direct unique fluorescent, biological or chemicalmolecules to individual segments in three or more component nanowireshas potential for further advances in gene/drug delivery, chemicalsensing and self-assembly.

It is understood that the above-described embodiments are illustrativeof only a few of the many possible specific embodiments, which canrepresent applications of the invention. Numerous and varied otherarrangements can be made by those skilled in the art without departingfrom the spirit and scope of the invention.

1-20. (canceled)
 21. A device for delivering biological molecules to acell comprising: a nanostructure comprising a plurality of discreteregions of respectively different materials; and a plurality ofmolecules of different composition bound to the respectively differentdiscrete regions.
 22. The device of claim 21 wherein the nanostructurecomprises a nanotube or nanowire having a plurality of axial segments ofrespectively different materials.
 23. The device of claim 21 wherein thematerials comprise different metals.
 24. The device of claim 21 whereinthe molecules include at least one material from the group consisting ofDNA, RNA, proteins, antigens, adjuvants endosomylytic agents andcytokines.
 25. The device of claim 21 wherein the different materialsinclude at least one material from the group consisting of metals andsemiconductors.
 26. The device of claim 21 wherein at least one of thedifferent materials comprises a material selected from the groupconsisting of nickel, gold and platinum.
 27. The device of claim 21wherein at least one of the molecules is bound by a linkage from thegroup consisting of thiol linkages, isonitrile linkages and carboxylatelinkages.
 28. The device of claim 21 wherein at least one of themolecules comprises compacted DNA.